The long term objective of this work is to understand the kinetic mechanisms by which normal blood vessels mount an appropriately timed and appropriately limited response during crisis or signalling events. Cell surface enzymes which interconvert agonists in the pericellular space play a significant role in this regulation. We propose to determine the kinetic properties which regulate the extracellular conversion of ATP to ADP, AMP, and adenosine on pig arterial smooth muscle cells and coronary microvascular endothelial cells. Synthesis and export of cAMP will be determined as a measure of cellular response to extracellular nucleotide conversion to adenosine. The specific aims for this project period are: 1) Determine whether smooth muscle cells have two 5'-nucleotidases, one similar to the endothelial enzyme and one different from it, by comparison of simulated progress of reaction curves with data obtained with cultured cells, by studying the effects of specific inhibitors of 5'-nucleotidase activity, and by determining immunologically whether the enzyme on endothelial cells is identical to some or all of the activity on smooth muscle cells. 2) Determine whether extracellular adenosine production and the adenosine-induced rise in cAMP are regulated differently on dividing smooth muscle cells and quiescent ones. Cell culture procedures will be adopted which maximize the expression of the quiescent phenotype. Freshly collected smooth muscle cells which still retain their contractility will be tested as well. 3) Determine whether on smooth muscle cells, adenosine delivery to its receptor is coupled to 5'-nucleotidase, and/or ATP or ADP potentiate the response to adenosine, using adenosine analogs and hydrolysis-resistant nucleotide analogs, and kinetic analysis of the rates of adenosine production and cAMP efflux. 4) Develop cultures of microvascular endothelial cells from pig heart and compare the regulation of ectonucleotidase kinetics and adenosine-stimulated cAMP release on these cells with pig aortic endothelial and smooth muscle cells. 5) Develop a kinetic model which includes both ectonucleotidase kinetics and the fluid dynamics of single pass perfusion. Transient high concentrations of extracellular adenine nucleotides occur during thrombus formation and neurotransmission, and they and the adenosine produced from them affect both platelet aggregation and smooth muscle tension in complex ways. The experiments proposed here should provide important information for understanding the control of the time courses of thrombus formation, vasoconstriction and vasodilation.